Acquisition et régulation des fonctions effectrices des lymphocytes T dans les maladies inflammatoires de l’intestin

L’intestin représente un microenvironnement complexe notamment par la présence du microbiote intestinal nécessitant un système immunitaire spécialisé incluant les cellules CD8 T résidente (Trm). Notre but est d’étudier la différentiation et la fonction de ces Trm dans la muqueuse intestinale. Nous souhaitons également identifier l’implication des Trm dans la maladie de Crohn (MC).Des cellules T provenant de sang ont été exposées in vitro dans conditions proches de la muqueuse pour étudier leur différentiation en Trm. L’environnement intestinal est capable de convertir des T CD8 du sang en un phénotype proche de celui de la muqueuse, notamment par l’acquisition de l’intégrine CD103. L'expression mutuellement exclusive de CD103 et KLRG1 sur les CD8 Trm semble définir des sous-populations fonctionnellement distinctes.Les Trm de patients ont été restimulé pour analyser leurs fonctions. Les CD103+ Trm sont plus sensibles à une restimulation TCR, mais les KLRG1+CD8 Trm expriment le Granzyme B sont augmentés dans la muqueuse des patients. Le transcriptome des CD103+ CD8 Trm est considérablement modifié dans la MC et montre une expression de gène associés à des signaux de danger, de réparation tissulaire et de recrutement lymphocytaire comparé aux individus contrôles. En parallèle, Nous avons établi un modèle de coculture d’organoide et de cellules T autologues. Notre but est d'étudier l’interaction et l'effet des ces cellules T sur des cellules épithéliales et de tester l’efficacité des biothérapies ciblant ces interactions comme des anticorps bloquant CD103 ou NKG2D, dont le développement est en cours dans la MC.The intestine is a complex microenvironment that requires an immune system with specific features to maintain homeostasis. Tissue resident memory (Trm) CD8 T cells from the intestinal tissue participate to this regulation. We aimed to study the differentiation and function of human CD8 Trm cells in the intestinal mucosa and their impact on inflammatory disorders such as Crohn’s disease (CD). We tested in vitro the acquisition of a mucosa-associated phenotype, by exposing blood T cells to cytokines mimicking the intestinal microenvironment. This stimulation converted activated blood CD8 T cells to a mucosal-like phenotype, mainly by acquisition of the tissue resident marker, integrin CD103.Blood and mucosal CD8 T cells isolated from CD patients and controls were characterized by flow cytometry to determine the specificities of intestinal Trm cells. Interestingly, the expression of KLRG1 and CD103, both receptor of E-cadherin expressed by epithelial cells, was mutually exclusive. Restimulated Trm cells in vitro showed that CD103 CD8 Trm cells were more responsive to TCR stimulation, while KLRG1 CD8 T cells displayed higher expression of cytotoxic molecules such as granzyme B. These results suggest that these markers define distinct functional Trm subsets.We analysed the transcriptome of sorted Trm subsets from inflammatory or control tissues and showed that CD8 Trm cells expressing CD103 had increase expression of cytokines and chemokines compared to other Trm cells. Additionally, CD103 expressing Trm cells from CD patients showed major transcriptomic differences compared to controls, with increase expression of genes involved in tissue repair and recruitment of immune effector cells. Taken together, these results suggest that Trm cells in the intestine are heterogeneous, as CD103 expressing cells display functions associated with alarm signals and tissue repair, while KLRG1 positive cells exhibit cytotoxic potential. To study the interactions of these T cells with intestinal epithelial cells, we have established intestinal epithelial organoid cultures with mucosal T cells. Our aims are to examine the molecules involved in lympho-epithelial interactions and study their functional consequences. To this end we will test and study the mechanisms of action of blocking antibodies targeting CD103 and NKG2D that are two pathways tested for the treatment of CD

Prediction of lipomatous soft tissue malignancy on MRI: comparison between machine learning applied to radiomics and deep learning

International audienceAbstract Objectives Malignancy of lipomatous soft-tissue tumours diagnosis is suspected on magnetic resonance imaging (MRI) and requires a biopsy. The aim of this study is to compare the performances of MRI radiomic machine learning (ML) analysis with deep learning (DL) to predict malignancy in patients with lipomas oratypical lipomatous tumours. Methods Cohort include 145 patients affected by lipomatous soft tissue tumours with histology and fat-suppressed gadolinium contrast-enhanced T1-weighted MRI pulse sequence. Images were collected between 2010 and 2019 over 78 centres with non-uniform protocols (three different magnetic field strengths (1.0, 1.5 and 3.0 T) on 16 MR systems commercialised by four vendors (General Electric, Siemens, Philips, Toshiba)). Two approaches have been compared: (i) ML from radiomic features with and without batch correction; and (ii) DL from images. Performances were assessed using 10 cross-validation folds from a test set and next in external validation data. Results The best DL model was obtained using ResNet50 (resulting into an area under the curve (AUC) of 0.87 ± 0.11 (95% CI 0.65−1). For ML/radiomics, performances reached AUCs equal to 0.83 ± 0.12 (95% CI 0.59−1) and 0.99 ± 0.02 (95% CI 0.95−1) on test cohort using gradient boosting without and with batch effect correction, respectively. On the external cohort, the AUC of the gradient boosting model was equal to 0.80 and for an optimised decision threshold sensitivity and specificity were equal to 100% and 32% respectively. Conclusions In this context of limited observations, batch-effect corrected ML/radiomics approaches outperformed DL-based models

Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment

Abstract Background Immunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues. Methods We analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro. Results We showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes. Conclusions Altogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A

T cell clonal expansions in ileal Crohn’s disease are associated with smoking behaviour and postoperative recurrence

T cell clonal expansions are present in the inflamed mucosa of patients with Crohn's disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection.Methods T cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >iO. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively.Results TCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment.Conclusion Clonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven

Identification of gene expression profiles associated with an Increased risk of post-operative recurrence in Crohn's disease

International audienceBACKGROUND AND AIMS: Ileocolonic resection is frequently needed in the course of Crohn's disease [CD] treatment and post-operative recurrence is extremely common. Our main objective was to analyse gene expression in the mucosa of CD patients at the time of surgery and at post-operative endoscopy, in order to identify predictors and mechanisms of early endoscopic recurrence. METHODS: We conducted transcriptome analyses on ileal mucosa samples collected from inflamed sections of the surgical specimens [n = 200], from ileal resection margins [n = 149] and in the neo-terminal ileum 6 months after surgery [n = 122]; these were compared with non-inflammatory bowel disease controls [n = 25]. The primary endpoint was post-operative endoscopic recurrence at 6 months. We applied regression models to identify gene signatures predicting endoscopic recurrence. RESULTS: Chronic inflammation was associated with strong expression of inflammatory genes [IL-6, IL-8, IL-1B] and decreased expression of genes involved in metabolic processes, but with a high inter-individual heterogeneity. Gene signatures associated with early endoscopic recurrence were mainly characterized by upregulation of TNFα, IFNγ, IL23A and IL17A. Pathway analyses showed that upregulation of mitochondrial dysfunction within the inflamed sections and JAK/STAT at the ileal margin were predictive of post-operative recurrence. A combined model integrating these top pathway signatures improved the prediction of endoscopic recurrence [area under the curve of 0.79]. STAT3 phosphorylation at the surgical ileal margin was associated with severe recurrence at 6 months. CONCLUSION: We identified several biological pathways in surgical ileal mucosa specimens associated with an increased risk of disease recurrence. Integration of the JAK/STAT and mitochondrial dysfunction pathways in the clinical model improved the prediction of post-operative recurrence

Association Between Microscopic Lesions at Ileal Resection Margin and Recurrence After Surgery in Patients With Crohn’s Disease

International audienceBackground and AimsDifferent types of histologic lesions at the ileal margin, detected by histology, have been associated with increased rates of recurrence after ileocaecal surgery in patients with Crohn’s disease (CD). We aimed to characterize histologic features of the ileal margin and to evaluate their association with disease recurrence.MethodsWe collected histologic data from 211 patients with ileal or ileocolonic CD who underwent ileocolonic resections at hospitals in France from September 2010 through December 2016. Ileal margins were analyzed. Early endoscopic recurrence was defined by a Rutgeerts score of i2 or more, 6 months after surgery. We also collected data from 10 adults with healthy ileum who underwent ileocecal resection for colonic tumors (controls). Clinical relapse was defined by CD-related symptoms confirmed by imaging, endoscopy, therapy intensification, CD-related complication, or subsequent surgery.ResultsSix months after surgery, 49% of patients had endoscopic recurrence; 5 years after surgery, 57% of patients had clinical relapse. Ileal margins were macroscopically affected in 20.9% of patients. CD transmural lesions at the margin (defined by mucosal ulceration or cryptitis, submucosal fibrosis and lymphoplasmacytic infiltrate of the subserosa) were observed in 13.6% of patients. Endoscopic recurrence was observed in 75% of patients with CD transmural lesions vs 46% of patients without (P =.005). In multivariate analysis, CD transmural lesions at the margin were independently associated with early endoscopic recurrence (OR, 3.83; 95% CI, 1.47-11.05; P =.008) and clinical recurrence (OR 2.04; 95% CI, 1.09-3.99; P =.026).ConclusionIn patients with CD, transmural lesions at the ileal margin were associated with an increased risk of post-operative recurrence. Histologic features of the ileal margin should be included in making decisions about post-operative therapy